Phenylalanine ammonia-lyase (PAL) is a non-mammalian enzyme widely distributed in plants (Koukol et al., J Biol Chem 236:2692-2698, 1961; Hanson et al., The Enzymes 7:75-166, 1972; Poppe et al., Curr Org Chem 7:1297-1315, 2003), some fungi (Rao et al., Can J Biochem 45(12):1863-1872, 1967; Abell et al., Methods Enzymol 142:242-253, 1987), and bacteria (Bezanson et al., Can J Microbiol 16:147-151, 1970; Xiang et al., J Biol Chem 277:32505-32509, 2002; Hill et al., Chem Commun 1358-1359, 2003) and can be recombinantly produced in Escherichia coli. 
PAL from two cyanobacteria strains, Anabaena variabilis (Av) and Nostoc punctiforme (Np), has been cloned and expressed in bacteria (e.g., Escherichia coli (E. coli), and was shown to display PAL enzyme activity in vitro and in vivo (see, e.g., U.S. Pat. Nos. 7,531,341; 7,534,595; 7,537,923 and 7,560,263). A pegylated recombinant Anabaena variabilis PAL (rAvPAL-PEG) has also been produced, wherein the rAvPAL protein was derivatized by covalent attachment of polyethylene glycol (PEG) to increase its half-life and optimize its pharmacokinetic profile and/or reduce its immunogenicity (Id.). rAvPAL-PEG has been shown to metabolize phenylalanine and is being developed as an enzyme substitution therapy (EST) for patients disorders or diseases associated with elevated levels of phenylalanine, such as PKU, as well as in cancer therapy (Id.).
A concern of administration of enzymes to patients, for example, for PKU EST or cancer therapy, is whether sufficient amounts of the enzyme are available in the body to exert a therapeutic effect in the patient. Moreover, in the case of pegylated enzymes, current methods used to determine the concentration of PEG are relatively insensitive (see, e.g., U.S. Pat. No. 6,596,849), and methods to detect the enzyme itself are complicated by the presence of the PEG. To date, such methods have not determined an effective way to expose the immunogenic epitopes of the therapeutic enzyme (e.g., rAvPAL-PEG) for capture by an immobilized antibody specific for the target therapeutic enzyme, and maximize the number of epitopes recognized by the antibodies, while at the same time minimizing the number of epitopes masked by the PEG molecules.
A further concern of administration of enzymes to a patient, e.g., for PKU EST or cancer therapy, is the possible development of enzyme-specific antibodies in patients, for example, those receiving multiple rounds of therapy. These enzyme-specific antibodies may precipitate potential adverse events and lead to changes in clinical efficacy, including, for example, anaphylactoid-type reactions associated with antibodies of the IgE isotype, changes in pharmacokinetic profile, neutralization of the enzymatic activity, interference with receptor-mediated enzyme uptake, and breaking of tolerance toward self proteins. Current assays to measure the amount of anti-enzyme antibodies in body fluids also suffer from a variety of technical issues, thereby making their interpretation difficult (see Mire-Sluis et al., J. Immunological Methods 289:1-16, 2004).
Given these concerns, as well as the shortcomings of currently available assays, there remains a need for a reliable, sensitive and specific assays to accurately detect or otherwise measure (i) an enzyme (e.g., a rAvPAL) in body fluids, tissues or other samples of a patient receiving the enzyme, e.g., for EST or cancer therapy; (ii) a pegylated enzyme, such as extensively pegylated enzyme (i.e., an enzyme having a sufficient number of PEG molecules attached such that at least some of the immunogenic epitopes of the enzyme are masked by the PEG molecules), (e.g., a rAvPAL-PEG) in bodily fluids, tissues or other samples of patient receiving the enzyme, e.g., for EST or cancer therapy; and/or (iii) enzyme- or PEG-specific antibodies (e.g., anti-rAvPAL, anti-rAvPAL-PEG antibodies, or anti-PEG antibodies) in body fluids, tissues or other samples of patient receiving the enzyme, e.g., for EST or cancer therapy. Such assays could enable assessment of the treatment regimen in a patient receiving the enzyme, e.g., for EST or cancer therapy and facilitate more efficient design of patient therapy.